| 1. BackgroundPseudomonas aeruginosa (P.aeruginosa) is a common gram-negative pathogen of nosocomial infection.It can be found in natural enviroment,skin of human body, respiratory tract et al.It is an important opportunistic nosocomial pathogen and especially in immuno-compromised patients.Recently, antibiotics such as?-lactams, glucocorticoids and immuno-suppressive drugs are used widely in clinical practice, the situations of resistance caused by P.aeruginosa become more serious even multidrug-resistant Pseudomonas aeruginosa (MDRPA) and pandrug-resistant Pseudomonas aeruginosa (PDRPA)appeared. The rising trend brings a great clinical challenge.Carbapenems are commonly used in severe infection caused by gram-negative bacterium, and effective in treatment of ESBLs and AmpC producing bacteria. However the carbapenem resistance is increasing and has become a significant problem in clinical P.aeruginosa infection.The mechanisms of carbapenem resistance in P.aeruginosa include production of (3-lactamases, loss of outer membran porin OprD2, and overproducing of active efflux pumps.In this study, a total of 645 non-duplicate P.aeruginosa isolates were collected from 28 hospitals in 16 different regions of China during the period from July 2006 to July 2007. The molecular epidemiology and resistance mechanisms of carbapenem-resistant P.aeruginosa isolates were studied.2.Materials and methodsThe 645 P.aeruginosa isolates collected from 28 hospitals during the period from July 2006 to July 2007.The MICs of imipenem and meropenem were determined by the agar dilution,and susceptibility to other nine antibiotics was determined by Kirby-Bauer method. Homology of carbapenem-resistant strains was analyzed by pulsed field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST). Metallo-beta-lactamases (MBLs) were detected by Etest. Polymerase chain reaction (PCR) amplification of the main?-lactamase genes incuding ESBLs, carbapenemases, and plasmid-mediated ampC genes.Quantification of the expression of MexAB-OprM,OprD2 and AmpC by real-time reverse transcriptase polymerase chain reaction(real-time RT PCR).The nucleotide sequences of the OprDi genes were amplified by PCR and sequenced. Categorical variables were compared using square test.3.ResultsOf the 645 P. aeruginosa isolates,245 (38.0%) isolates were resistant to imipenem and 184 (28.5%) isolates were resistant to meropenem,227(88.0%) isolates were multidrug-resistant, and 11(4.3%) isolates were panresistant. The 258 carbapenemresistant strains (imipenem-and/or meropenem-resistant) were chosen for further study.PFGE revealed remarkable clonal diversity, since 89 different clones were identified among 246 isolates; the other 12 isolates could not be typed by PFGE. A total of 64 sequnce types (STs) were detected by MLST, of which 55 STs were newly found.Among the 258 carbapenem-resistant P. aeruginosa isolates collected throughout China?22 MBL-positive strains were confirmed by PCR and DNA sequence analysis.The blaOXA-50 gene was detected among all of the P.aeruginosa isolates in this study. No other carbapenemase or plasmid-mediated AmpC genes were detected among the 258 carbapenem-resistant P. aeruginosa isolates.Nineteen MBL producing isolates were detected by MBL Etest. The other three isolates, zy8 (IMP-1 gene positive), tj22 (VIM-2 gene positive) and ss37 (VIM-2 gene positive), could not be detected by MBL Etest.The association of AmpC hyperproduction with meropenem resistance was explored. Only 5.4%(14/258) of the carbapenemresistant isolates in this study were found to hyperproduce AmpC.In this study,79.8%(206/258) of the carbapenem-resistant isolates showed decreased transcription of oprD2 to variable degrees; in 61.2%(158/258) of the isolates the reduction was significant (<0.4-fold compared with P. aeruginosa PAO1) and in 19 isolates (7.4%) oprDo transcription was zero or very low (<0.01 compared with P. aeruginosa PAO1).46.1%(119/258) of the carbapenem-resistant isolates showed mexA levels substantially higher (>2.5-fold) compared with those in the reference strain PAO1.4.ConclusionsThe resistance rate to carbapenems of P. aeruginosa strains in China was high. The molecular epidemiology studies, through PFGE and MLST, revealed remarkable clonal diversity of the carbapenem-resistant P.aeruginosa isolates in China. The interhospital transmission of carbapenem-resistant strains was apparently limited.MBLs producing and AmpC hyperproducing were not the main carbapenem resistance mechanisms of P. aeruginosa isolates in China.The decreased oprD2 transcription and increased transcription of the efflux genes were the main carbapenem resistance mechanism of clinical P. aeruginosa isolates in China. However, it does not fully explain the resistance patterns observed in some isolates investigated.The CTX-M-13,14,15, and 3 genotypes of ESBLs were firstly discribed in P. aeruginosa isolates. |
PDF Full Text Request